Nitration of tyrosine is an important pathological marker of peroxynitrite and other reactive nitrogen species in a wide range of human diseases. A number of environmental toxins also induce tyrosine nitration in their target tissues. Using antibodies first developed in the Project leader?s lab, over 700 studies have been published in the past 5 years on tyrosine nitration in at least 80 different disease states. Approximately 20 different nitrated proteins have been identified and many sites of nitration have been identified by mass spectrometry. Some of the most susceptible proteins to nitration are the disassembled subunits of structural proteins. Although many aspects of tyrosine nitration are controversial and still poorly understood, the investigators have confirmed by multiple methodologies reports from other groups that 1 to 3% of total tyrosine in proteins can be nitrated in diseased human lung, heart and other tissues. The long-term goal of the Project leader's laboratory is to understand the causes and functional significance of tyrosine nitration in biological systems. The central hypothesis of the proposed work is that nitration is a general mechanism to disrupt the interactions between structural and regulatory proteins that may be a resistance mechanism against viral infections. A major limitation in the studies of tyrosine nitration remains the limited affinity and specificity of antibodies to nitrotyrosine. To improve upon existing antibodies, the investigators are generating recombinant antibodies using phage display methods to specific sequences containing nitrotyrosine. One advantage of this approach is the identification of short protein sequences from the antibody's recognition domain that preferentially bind nitrotyrosine. The first aim is to increase the affinity of these phage display antibodies through directed evolution and to identify protein sequences that bind nitrotyrosine. The second aim is to characterize what makes certain sequences in structural proteins more susceptible to nitration and characterize the functional consequences of nitration on assembly. The third aim is to characterize why urate is a superior inhibitor of tyrosine nitration than ascorbate or thiols when cells are exposed to peroxynitrite. This surprising result can be used to provide important mechanistic insight into the role of tyrosine nitration versus thiol oxidation in peroxynitrite toxicity.